US20240191205A1 - Efficient and non-genetically modified ipsc-induced, industrialized single clone selection platform, and use - Google Patents

Efficient and non-genetically modified ipsc-induced, industrialized single clone selection platform, and use Download PDF

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US20240191205A1
US20240191205A1 US18/512,843 US202318512843A US2024191205A1 US 20240191205 A1 US20240191205 A1 US 20240191205A1 US 202318512843 A US202318512843 A US 202318512843A US 2024191205 A1 US2024191205 A1 US 2024191205A1
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Lida WU
Yuchun GU
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Allife Medicine Zhuhai Ltd
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Definitions

  • the present invention belongs to the field of biomedical technology and relates to an efficient and non-genetically modified iPSC induced, industrialized monoclone selection platform, and its application.
  • Takahashi and Yamanaka introduced several transcription factors into differentiated mouse skin fibroblasts and obtained pluripotent stem cells similar to embryonic stem cells (ESCs), known as “induced pluripotent stem cells (iPSCs)” (Takahashi K, Yamanaka S Introduction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors [J] Cell, 2006, 126 (4): 663-676).
  • iPSCs embryonic stem cells
  • Takahashi successfully obtained human iPSCs using human fibroblasts (Takahashi K, Tanabe K, Ohnuki M, et al. Introduction of pluripotent stem cells from adult human fibroblasts by defined factors [J]. Cell, 2007, 131 (5): 861-872.).
  • iPSC not only overcomes ethical controversies and other issues in human ESCs research, but also avoids the problem of lacking human oocytes in nuclear transfer technology. It provides an important research platform for the mechanism of disease occurrence and development, developmental biology research, gene and protein function research, and drug screening research and development. At the same time, it also provides a source of seed cells for the clinical application of regenerative medicine and stem cells.
  • the clinical applications of iPSCs can be divided into three categories: disease models (for studying disease mechanisms and rare disease drug screening), stem cell therapy, and tissue and organ regeneration.
  • iPSCs are induced to differentiate into functional three-dimensional tissues or organs by adding specific biophysical and biochemical inducing factors to the appropriate extracellular micro-environment.
  • iPSC induction methods for iPSC can be divided into integrated and non-integrated types.
  • integrated reprogramming often utilizes retroviruses, lentivirus carriers, and other methods to achieve gene introduction, integration, and reprogramming.
  • retroviruses lentivirus carriers
  • other methods to achieve gene introduction, integration, and reprogramming.
  • this method is efficient, the integration of exogenous viral DNA into the host cell genome is highly likely to lead to abnormal host gene expression, resulting in unstable iPSCs, great potential for cancer transformation, and lack of safety.
  • Non-integrated reprogramming reduces changes in chromosome structure and to some extent reduces the possibility of gene mutations and carcinogenesis, such as adenoviruses, Sendai viruses, retroviruses, transposons, plasmids, microcyclic DNA, recombinant proteins, small molecule compounds, RNAs, and other methods that can produce iPSCs.
  • Norikatsu et al. reprogrammed mouse and human cells into inducing pluripotent stem cells using mature double stranded RNA miR-200c combined with the mir-369 and miR-302 families (Miyoshi N, Ishii H, Nagano H, et al.
  • the present invention provides an efficient and non-genetically modified iPSC induction, industrial monoclonal selection platform, which can efficiently reprogram and only require the use of the minimum number of reprogramming factors (OCT4, SOX2, E6, E7).
  • OCT4, SOX2, E6, E7 the minimum number of reprogramming factors
  • the present invention uses SSEA4, TRA-1-60 as screening markers and uses flow cytometry sorting technology, sorting individual cells with positive characterization onto a 96 well plate can easily obtain a large number of single cell clones with minimal manual operation, which can be used for industrial production.
  • the first aspect of the present invention provides a reprogramming factor combination for reprogramming somatic cells into inducing pluripotent stem cells.
  • the reprogramming factor combination is selected from OCT4, SOX2, E6, E7, KLF4, L-MYC, LIN28, NANOG, and SV40LT.
  • the reprogramming factor combination is OCT4, SOX2, E6, E7.
  • the form of the reprogramming factor combination includes DNA, RNA, and protein.
  • the DNA is cDNA and the RNA is mRNA.
  • induced pluripotent stem cells used in the application are interchangeable and refer to a type of pluripotent stem cell that has the ability to self-renew and differentiate into three embryonic layers by artificially reprogramming non pluripotent cells (such as somatic cells).
  • Reprogramming refers to a process of obtaining inducing pluripotent stem cells through exogenous genes expression, compound induction, epigenetic modification and other pathways
  • the second aspect of the present invention provides a reprogramming factor carrier for reprogramming somatic cells into inducing pluripotent stem cells.
  • the reprogramming factor carrier comprises a reprogramming factor carrier obtained by encoding the reprogramming factor described in the first aspect of the present invention in one and/or more skeleton carriers.
  • the skeleton carrier includes pEGFP N1, pCMVp-NEO-BAN, PEGFP, PEGFT actin, pSV2, and CMV4.
  • the skeleton carrier is pEGFP N1.
  • the reprogramming factor carrier includes one and/or more of the following reprogramming factor carriers: carriers encoding OCT4, carriers encoding SOX2, carriers encoding E6, carriers encoding E7, carriers encoding OCT4 and SOX2, carriers encoding OCT4 and E6, carriers encoding OCT4 and E7, carriers encoding SOX2 and E6, carriers encoding SOX2 and E7, carriers encoding E6 and E7, carriers encoding OCT4 and SOX2 and E6 Carriers encoding OCT4 and SOX2 and E7, carriers encoding OCT4 and E6 and E7, carriers encoding SOX2 and E6 and E7, carriers encoding OCT4 and SOX2 and E6 and E7.
  • the reprogramming factor carrier includes two types of reprogramming factor carriers: carriers encoding OCT4 and SOX2, and carriers encoding E6 and E7.
  • OCT4 and SOX2 in the carrier encoding OCT4 and SOX2 are connected through a spacer sequence and/or directly connected.
  • OCT4 and SOX2 in the carrier encoding OCT4 and SOX2 are connected through a spacer sequence.
  • the spacer sequence includes IRES and/or self cleaving peptide 2A.
  • the self cleaving peptide 2A is selected from T2A, P2A, E2A, F2A.
  • the spacer sequence is IRES.
  • connection method of OCT4 and SOX2 in the carrier encoding OCT4 and SOX2 includes: OCT4, IRES, SOX2 sequentially connected in series, SOX2, IRES, OCT4 sequentially connected in series.
  • connection mode of OCT4 and SOX2 in the carrier encoding OCT4 and SOX2 is that OCT4, IRES, and SOX2 are sequentially connected in series.
  • E6 and E7 in the carriers encoding E6 and E7 are connected through spacer sequence and/or directly connected.
  • E6 and E7 in the carriers encoding E6 and E7 are connected through spacer sequences.
  • the spacer sequence includes IRES, the self cleaving peptide 2A, and/or any two nucleotides.
  • the self cleaving peptide 2A is selected from T2A, P2A, E2A, F2A.
  • any two nucleotides are selected from AA, TT, CC, GG, AT, TA, AC, CA, AG, GA, TC, CT, TG, GT, CG, GC.
  • interval sequence is IRES
  • connection method of E6 and E7 in the carrier encoding E6 and E7 includes: E6, IRES, E7 sequentially connected in series, E7, IRES, E6 sequentially connected in series.
  • connection mode of E6 and E7 in the carrier encoding E6 and E7 is sequential series connection of E6, IRES, and E7.
  • nucleotide sequences of the carriers encoding OCT4 and SOX2 are shown in SEQ ID NO: 1.
  • nucleotide sequences of the carriers encoding E6 and E7 are shown in SEQ ID NO: 2.
  • IRES refers to the internal ribosome entry site, which is a cis acting RNA sequence that can mediate the internal entry of 40S ribosomal subunits on certain eukaryotic cells and viral messenger RNA upstream of the translation start codon. The presence of IRES ensures the co expression of multiple genes under the control of the same promoter.
  • the third aspect of the present invention provides a preparation method for inducing pluripotent stem cells derived from somatic cells.
  • the method comprises the following steps: delivering the reprogramming factor combination described in the first aspect of the present invention to somatic cells.
  • the delivery of the reprogramming factor combination is achieved by introducing the reprogramming factor carrier into somatic cells.
  • the methods of introduction include electro transfection, microinjection, gene gun, DEAE glucan, calcium phosphate co precipitation transfection, and artificial liposome methods.
  • the method of introduction is an electro transfection method.
  • the reprogramming factor carrier is the reprogramming factor carrier described in the second aspect of the present invention.
  • the dosage of the reprogramming factor carrier is: to 80 to 2000,000 somatic cells/100 ⁇ L, added 0.5-8 ⁇ g carriers encoding OCT4 and SOX2 and carriers encoding E6 and E7.
  • culture somatic cells obtained by introducing reprogramming factor carriers.
  • somatic cells obtained by introducing reprogramming factor carriers are cultured in a cell culture plate.
  • the cell culture plate is a culture plate coated with extracellular matrix proteins.
  • the extracellular matrix protein includes laminin.
  • the laminin is human laminin 521.
  • the cell culture plate also includes hematopoietic stem cells.
  • the E8 medium is used to continue cultivation for 2-4 days for full medium change.
  • the E8 medium is used to continue cultivation until a large number of clones are formed.
  • the obtained clones are screened, labeled, and sorted to obtain somatic derived induced pluripotent stem cells.
  • the screening markers include SSEA4, TRA-1-60, SSEA1, TRA-1-81, SSEA3, OCT4, SOX2, and NANOG.
  • the screening markers are selected from SSEA4, TRA-1-60, SSEA1, TRA-1-81.
  • the screening markers are SSEA4, TRA-1-60.
  • the sorting is carried out using flow cytometry sorting technology.
  • the somatic cells include peripheral mononuclear blood cells, fibroblasts, umbilical cord blood cells, fibroblast like synovial cells, myocardial cells, liver cells, neural cells, hematopoietic stem cells, pancreatic islet cells, gastric epithelial cells, B lymphocytes, T lymphocytes, adipocytes, and pancreas ⁇ Cells, keratinocytes, mesenchymal stromal cells, epithelial cells, endothelial cells.
  • the somatic cells are peripheral monocytes.
  • the sorting process prior to the sorting process, it also includes treating cells with ROCK signaling pathway inhibitors and digesting cells.
  • the processing time is 2 hours.
  • the ROCK signaling pathway inhibitors include Thiazovivin, Belumosudil mesylate, SAR407899, BDP5290, Belumosudil (SLx-2119), ZINC00881524, HA 1100 hydrochloride, Ripaudil (K-115) dihydrorate, SR 3677, GSK180736A, Hydroxyfasudil hydrochloride, GSK26962A, Y-39983 dihydrochloride, Fasudil, GSK429286A, RKI-1447, Fasudil hydrochloride, Y-27632 hydrochloride.
  • the ROCK signaling pathway inhibitor is Thiazovivin.
  • the digestive fluid used for cell digestion includes TRYPLE, trypsin, chymotrypsin, and intestinal kinase.
  • the digestive solution is TRYPLE.
  • the method also includes culturing and subculturing SSEA4 and TRA-1-60 single positive or double positive cells obtained from sorting.
  • the culture involves seeding the selected SSEA4 and TRA-1-60 single or double positive cells onto a cell culture plate coated with human laminin-521, and culturing them in a 37° C., 5% CO2 incubator for 7-14 days, with daily full fluid exchange.
  • cell passage is carried out.
  • the fourth aspect of the present invention provides an induced pluripotent stem cell or cell population derived from somatic cells.
  • the induced pluripotent stem cells or cell populations are prepared using the method described in the third aspect of the present invention.
  • the fifth aspect of the present invention provides a reprogrammed somatic cell.
  • the reprogrammed somatic cells are cells obtained by introducing the reprogrammed factor carrier described in the second aspect of the present invention into somatic cells.
  • the methods of introduction include electro transfection, microinjection, gene gun, DEAE glucan, calcium phosphate co precipitation transfection, and artificial liposome methods.
  • the method of introduction is an electro transfection method.
  • the somatic cells include peripheral mononuclear blood cells, fibroblasts, umbilical cord blood cells, fibroblast like synovial cells, myocardial cells, liver cells, neural cells, hematopoietic stem cells, pancreatic islet cells, gastric epithelial cells, B lymphocytes, T lymphocytes, adipocytes, and pancreas ⁇ Cells, keratinocytes, mesenchymal stromal cells, epithelial cells, endothelial cells.
  • the “reprogrammed somatic cells” are reprogrammed intermediate state cells, which are in an incomplete reprogrammed cell state and are an intermediate state during the reprogramming process, including activation of some pluripotent genes that can be induced to become pluripotent stem cells under suitable culture conditions.
  • the sixth aspect of the present invention provides a culture medium for reprogramming somatic cells into inducing pluripotent stem cells.
  • the culture medium is supplemented with selected OCT4 protein, SOX2 protein, E6 protein, E7 protein, KLF4 protein, L-MYC protein, LIN28 protein, NANOG protein, and SV40LT protein.
  • the culture medium is supplemented with OCT4 protein, SOX2 protein, E6 protein, and E7 protein.
  • the seventh aspect of the present invention provides a kit for producing induced pluripotent stem cells.
  • the kit comprises a reprogramming factor combination as described in the first aspect of the present invention, a reprogramming factor carrier as described in the second aspect of the present invention, and/or reprogrammed somatic cells as described in the fifth aspect of the present invention.
  • the eighth aspect of the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises induced pluripotent stem cells or cell populations as described in the fourth aspect of the present invention.
  • the pharmaceutical composition also includes pharmaceutically acceptable carriers.
  • the ninth aspect of the present invention provides a method for inducing pluripotent stem cell monoclonal selection from somatic cells.
  • the method comprises the following steps:
  • step (1) further includes treating cells with ROCK signaling pathway inhibitors before digesting the cells.
  • the processing time is 2 hours.
  • the ROCK signaling pathway inhibitors include Thiazovivin, Belumosudil mesylate, SAR407899, BDP5290, Belumosudil (SLx-2119), ZINC00881524, HA 1100 hydrochloride, Ripsudil (K-115) dihydrorate, SR 3677, GSK180736A, Hydroxyfasudil hydrochloride, GSK26962A, Y-39983 dihydrochloride, Fasudil, GSK429286A, RKI-1447, Fasudil hydrochloride, Y-27632 hydrochloride.
  • the ROCK signaling pathway inhibitor is Thiazovivin.
  • the digestive fluid used for the digestive cells in step (1) includes TRYPLE, trypsin, chymotrypsin, and intestinal kinase.
  • the digestive solution used for the digestive cells in step (1) is TRYPLE.
  • the screening markers described in step (2) include SSEA4, TRA-1-60, SSEA1, TRA-1-81, SSEA3, OCT4, SOX2, and NANOG.
  • the screening markers described in step (2) are selected from SSEA4, TRA-1-60, SSEA1, and TRA-1-81.
  • the screening markers described in step (2) are SSEA4 and TRA-1-60.
  • the cell culture plate described in step (4) is a cell culture plate coated with extracellular matrix proteins.
  • the extracellular matrix protein includes laminin.
  • the laminin is human laminin 521.
  • the method also includes expanding the culture of cell clones.
  • the tenth aspect of the present invention provides applications in any of the following aspects:
  • the diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, Down's syndrome, X chromosome vulnerability syndrome, Reiter syndrome, Huntington's disease, familial autonomic nervous dysfunction disease, schizophrenia, ataxia, diabetes, cardiovascular disease, age-related macular degeneration, myopic macular degeneration, Steger's disease, kidney disease Liver disease, lung disease, hemophilia, myeloma.
  • Alzheimer's disease Parkinson's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, Down's syndrome, X chromosome vulnerability syndrome, Reiter syndrome, Huntington's disease, familial autonomic nervous dysfunction disease, schizophrenia, ataxia, diabetes, cardiovascular disease, age-related macular degeneration, myopic macular degeneration, Steger's disease, kidney disease Liver disease, lung disease, hemophilia, myeloma.
  • the tumor includes melanoma, prostate cancer, breast cancer, lung cancer, kidney cancer, liver cancer, cervical cancer, vulva cancer, B-cell lymphoma, T-cell lymphoma, myeloma, leukemia, hematopoietic system tumor, thymoma, lymphoma, sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, skin cancer, uterine cancer, endometrial cancer, adenocarcinoma, pancreatic cancer, colorectal cancer, anal cancer, bladder cancer, ovarian cancer Squamous cell carcinoma, basal cell carcinoma, brain cancer, angiosarcoma, vascular endothelial tumor, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular cancer, gastrointestinal cancer.
  • B-cell lymphoma T-cell lymphoma
  • myeloma myeloma
  • leukemia
  • precursor cells refers to all intermediate cells involved in the process of inducing pluripotent stem cells or cell populations, reprogrammed somatic cells to differentiate into differentiated cells.
  • the intermediate cells include all cells involved in the process, except for the initial cells (inducing pluripotent stem cells or cell populations, reprogrammed somatic cells) and terminal cells (differentiated cells).
  • the efficient and non-genetically modified iPSC induction and industrialized monoclonal selection platform reduces potential carcinogenicity by using the minimum number of reprogramming factors, while achieving high induction efficiency and improving the operability of reprogramming technology.
  • the present invention uses SSEA4/TRA-1-60 as screening markers and uses flow cytometry sorting technology, sorting individual cells with positive characterization onto a 96 well plate can easily obtain a large number of single cell clones with minimal manual operation, which can be used for industrial production.
  • FIG. 1 is the flowchart of an efficient and non-genetically modified iPSC induction and industrial monoclonal selection platform developed by the present invention, as well as the flowchart of the current iPSC induction method, in which a of FIG. 1 shows the flowchart of the current iPSC induction method, and b of FIG. 1 shows the flowchart of the efficient and non-genetically modified iPSC induction and industrial monoclonal selection platform developed by the present invention;
  • FIG. 2 shows the morphology of iPSC reprogramming using OCT4-IRES-SOX2 and E6-E7 plasmids after PBMC electro transfection on the first day;
  • FIG. 3 shows the morphology of epithelial cells produced on the 7th day after iPSC reprogramming using OCT4-IRES-SOX2 and E6-E7 plasmids;
  • FIG. 4 shows the morphology of epithelial cells produced on day 20 after iPSC reprogramming using OCT4-IRES-SOX2 and E6-E7 plasmids;
  • FIG. 5 shows the results of OSE6-IPSC alkaline phosphatase staining using OCT4-IRES-SOX2 and E6-E7 plasmids
  • FIG. 6 shows the OSE6-IPSC immunofluorescence results obtained using OCT4-IRES-SOX2 and E6-E7 plasmids
  • FIG. 7 shows the flow cytometry results of OSE6-IPSC obtained using the OCT4-IRES-SOX2 and E6-E7 plasmids, in which a of FIG. 7 shows NANOG, b of FIG. 7 shows OCT4, c of FIG. 7 shows SOX2, d of FIG. 7 shows SSEA1, e of FIG. 7 shows SSEA4, f of FIG. 7 shows TRA-1-60, and g of FIG. 7 shows TRA-1-81;
  • FIG. 8 shows the HE staining results of OSE6-IPSC teratoma slices obtained using OCT4-IRES-SOX2 and E6-E7 plasmids, where a represents the hair follicle structure, b represents the cartilage structure, and c represents the digestive tract epithelial structure;
  • FIG. 9 shows the results of OSE6-IPSC karyotype detection using OCT4-IRES-SOX2 and E6-E7 plasmids.
  • the pEGFP N1 plasmid described in this embodiment was purchased from Addgene Company.
  • PBMCs Peripheral blood mononuclear cells
  • StemSpanTM SFEM II is purchased from STEMCELL Technologies under item #09655
  • StemSpanTM The CD34+Expansion Supplement was purchased from STEMCELL Technologies under item #02691.
  • the hematopoietic stem cell basic culture medium group was composed by StemSpanTM SFEM II and StemSpanTM CD34+Expansion Supplement, configured according to the manufacturer's requirements;
  • the PBMC obtained after electric transfection in Example 2 of the present invention recombinant human laminin-521 was purchased from Thermo Fisher Scientific Co., Ltd., product number A29249; the E8 culture medium was purchased from Thermo Fisher Scientific Co., Ltd. with product number A1517001; TRYPLE was purchased from Thermo Fisher Scientific Co., Ltd. with product number 12604013; Alexa Fluor® 488 anti-human SSEA-4 Antibody is purchased from BioLegend Company with product number 330412; Alexa Fluor® The 594 anti-human TRA-1-60-R Antibody was purchased from Biogene Corporation under product number 330616.
  • Alkaline Phosphatase Staining Solution Alkaline phosphatase Alkaline phosphatase 3 mL 3.03 mL staining solution chromogenic buffer (AP staining solution) BCIP solution (300X) 10 ⁇ L NBT solution (150X) 20 ⁇ L
  • iPSCs were positively expressed by alkaline phosphatase staining (see FIG. 5 ), indicating high alkaline phosphatase activity in iPSCs and iPSCs is in their undifferentiated state.
  • the information of the primary and secondary antibodies used in this example is as follows: Anti Nanog antibody (Abcam company, product number: ab109250); Mouse anti SSEA-4 (Abcam Company, product number ab16287); Mouse anti TRA-1-60 (Abcam company, product number: ab16288); Anti Oct4 antibody (Abcam company, product number: ab19857); Recombinant Anti SOX2 antibody [EPR3131] (Abcam Company, ab92494); Goat anti Mouse IgG3 Cross Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, A-21151); Goat anti Mouse IgM (Heavy chain) Cross Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, A-21042); Donkey anti rabbit IgG (H+L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor 594 (Invitrogen, A-21207); Donkey anti rabbit IgG (H+L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, A-21
  • the antibodies used in this example are all purchased from Biogene Company, and the antibodies include: Alexa Fluor® 594 anti Nano Anti body (674204); Alexa Fluor® 594 anti Oct4 (Oct3) Anti body (653708); Alexa Fluor® 488 anti human SSEA-4 Anti body (330412); PE anti mouse/human CD15 (SSEA-1) Anti body (125605); Alexa Fluor® 488 anti human TRA-1-60-R Antibody (330613); Alexa Fluor® 488 anti human TRA-1-81 Anti body (330709); Alexa Fluor® 555 Mouse anti Sox2 (560293).
  • a is the hair follicle structure
  • cartilage structure is the cartilage structure
  • c is the digestive tract epithelial structure, indicating that the obtained iPSC can successfully form teratomas and has pluripotency for in vivo differentiation.
  • the karyotype detection results of OSE6-IPSC obtained using the OCT4-IRES-SOX2 and E6-E7 plasmids in this example are shown in FIG. 9 .
  • 46 chromosomes with normal numbers and XX sex chromosomes are visible, indicating a female karyotype and no abnormalities in chromosome structure. This indicates that the OSE6-IPSC obtained using the OCT4-IRES-SOX2 and E6-E7 plasmids does not exhibit chromosomal aberrations and all exhibit normal karyotypes.
  • the commercial reprogramming kit CD34+Progenitor Reprogramming Kit used in this example was purchased from STEMCELL Technologies under item #05925.

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